Deciphering the poly-ubiquitin code with isopeptide selective antibodies

用同肽选择性抗体破译多聚泛素密码

基本信息

批准号：
8714719
负责人：
Mark Mason
金额：
$ 22.37万
依托单位：
LIFESENSORS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-06 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8714719
关键词：
Active Sites Address Advertising Affect Affinity Alzheimer&apos s Disease Amino Acids Antibodies Antibody Affinity Antibody Specificity Antigens Apoptosis Architecture Autoimmune Process Autophagocytosis Binding Biochemistry Biological Biological Assay Biology C-terminal Cell Cycle Progression Cell physiology Cellular biology Chemicals Clinical Code Communicable Diseases Communities Complex Mixtures Coupled DNA Damage DNA Repair Detection Development Diagnostic Disease Distal Endocytosis Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Epitopes Excision Exhibits Figs - dietary Fluorescent Dyes Glycine Goals Grant Half-Life Human Genome Image Immune response Immunoglobulin Somatic Hypermutation Inflammation Label Length Libraries Link Lysine Malignant Neoplasms Marketing Measures Mediating Modification Molecular Weight Nature Nerve Degeneration Pathway interactions Phase Phospho-Specific Antibodies Phosphorylation Play Polymers Polyubiquitin Population Post-Translational Protein Processing Protein Chemistry Protein Microchips Proteins Proteomics Reagent Regulation Research Role Sepharose Signal Pathway Signal Transduction Site Small Business Innovation Research Grant Specificity Staining method Stains Surface Surface Plasmon Resonance System Techniques Technology Time UBD protein Ubiquitin Ubiquitin-Conjugating Enzymes Ubiquitination Western Blotting Work Yeasts amino group antigen binding base carboxylate cell growth regulation drug discovery experience innovation isopeptidase meetings multicatalytic endopeptidase complex protein degradation protein function protein structure function protein transport public health relevance receptor receptor recycling response synthetic peptide tool ubiquitin-protein ligase yeast genetics

项目摘要

DESCRIPTION (provided by applicant): Ubiquitin (Ub), a 76 amino acid protein, is implicated in protein degradation and a myriad of cell signaling pathways, including DNA damage response, protein trafficking, cell-cycle progression, inflammation, immune response and regulation of apoptosis. Ubiquitylation occurs through formation of an isopeptide bond between the C-terminus of Ub and the ?-amino group of a lysine (Lys) residue on target proteins. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can be further ubiquitylated, generating poly-Ub chains ranging from 2 to 7 Ubs in length. The ability of Ub to form polymers through various lysines appears to be central to the versatility of this system in regulating cell processes. Research in the ubiquitin field is hampered by a lack of antibodies or other tools that selectively recognize poly-ubiquitylated proteins. To address this issue we have combined our experience of generating isopeptide selective poly-Ubs and yeast genetics to generate diverse single chain antibodies that will advance the field of cell biology. I this grant we propose to use yeast display of single domain camelid antibodies to select for linkage specific binders. We will employ synthetic peptides which encompass both the isopeptide bond and the sequence surrounding the lysine attachment site in ubiquitin as well as a complete set of di-ubiquitins comprising each of the 7 isopeptide linkages. Both synthetic peptide antigens and diUbs will be used individually to pan for antibodies. LifeSensors and the scientific community will use these tools to chart new pathways in cell biology and biochemistry. These tools will be used as detection agents in protein chemistry, clinical diagnostic applications and imaging studies. These tools will unmask ubiquitomics of the cellular proteins and uncover new roles of unique poly-ubiquitylated proteins. Given the poor nature of tools available for K48- and K63- poly-Ub linkages, as a proof-of- principle, the phase I grant will focus on K48- and K63-linkages and expand to the remaining linkages in Phase II. We believe that development of linkage selective tools will have as great an impact on cell biology as the development of phospho-specific antibodies had on cellular phosphorylation.

描述（由申请人提供）：泛素 (Ub) 是一种 76 个氨基酸的蛋白质，参与蛋白质降解和多种细胞信号传导途径，包括 DNA 损伤反应、蛋白质运输、细胞周期进程、炎症、免疫反应和调节细胞凋亡。泛素化是通过 Ub C 末端和靶蛋白上赖氨酸 (Lys) 残基的 α-氨基之间形成异肽键而发生的。 Ub 本身有 7 个 Lys 残基（K6、K11、K27、K29、K33、K48 和 K63），每个残基都可以进一步泛素化，产生长度为 2 至 7 个 Ub 的多聚 Ub 链。 Ub 通过各种赖氨酸形成聚合物的能力似乎是该系统在调节细胞过程中的多功能性的核心。由于缺乏选择性识别多聚泛素化蛋白的抗体或其他工具，泛素领域的研究受到阻碍。为了解决这个问题，我们结合了生成异肽选择性多聚泛素和酵母遗传学的经验，生成了多种单链抗体，这将推动细胞生物学领域的发展。在这笔资助中，我们建议使用单域骆驼抗体的酵母展示来选择连接特异性结合物。我们将使用包含异肽键和泛素中赖氨酸附着位点周围序列的合成肽，以及包含 7 个异肽键中每一个的完整双泛素集。合成肽抗原和 diUb 将单独用于淘选抗体。 LifeSensors 和科学界将使用这些工具来绘制细胞生物学和生物化学的新途径。这些工具将用作蛋白质化学、临床诊断应用中的检测剂和影像学研究。这些工具将揭示细胞蛋白质的泛素组学，并揭示独特的多泛素化蛋白质的新作用。鉴于可用于 K48- 和 K63- 聚 Ub 连接的工具性质较差，作为原理证明，第一阶段拨款将重点关注 K48- 和 K63- 连接，并在第二阶段扩展到其余连接。我们相信，连锁选择性工具的发展将对细胞生物学产生巨大的影响，就像磷酸化特异性抗体的发展对细胞磷酸化产生的影响一样。